Download MagJET Whole Blood gDNA Kit

PRODUCT INFORMATION
Thermo Scientific
MagJET Whole Blood Genomic DNA Kit
#K2741, #K2742
Read Storage information (p. 4) upon receipt and store the kit
components appropriately!
www.thermoscientific.com/onebio
#__
Lot 00000000
Expiry Date 00.0000
CERTIFICATE OF ANALYSIS
Thermo Scientific™ MagJET™ Whole Blood Genomic DNA Kit is qualified by isolating
genomic DNA from 200 μL of frozen human whole blood following the protocol outlined in the
manual. The quality of isolated DNA is evaluated spectrophotometrically and by agarose gel
electrophoresis. The purified DNA has an A260/A280 ratio of 1.8±0.2. The functional quality of
purified DNA is evaluated by digestion with restriction endonucleases.
Quality authorized by:
Rev.1
Jurgita Žilinskienė
cc
2
Contents
page
COMPONENTS OF THE KIT................................................................................................................ 4
STORAGE ............................................................................................................................................ 4
DESCRIPTION ..................................................................................................................................... 4
PRINCIPLE ........................................................................................................................................... 4
IMPORTANT NOTES ........................................................................................................................... 5
ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED ................................................................. 5
PROTOCOL SELECTION GUIDE ........................................................................................................ 6
GENOMIC DNA PURIFICATION PROTOCOLS AND PIPETTING INSTRUCTIONS ........................... 7
Protocol A. Instructions for genomic DNA purification from 200 μL of whole blood using KingFisher
Flex 96 and Microtiter deep well 96 plates ............................................................................................... 7
Protocol B. Instructions for genomic DNA purification from 1 mL of whole blood using KingFisher
Flex and 24 deep well plates. .................................................................................................................... 9
Protocol C. Instructions for DNA purification from 2 mL of whole blood using KingFisher Flex and
24 deep well plates ....................................................................................................................................11
Protocol D. Instructions for genomic DNA purification from 200 μL of whole blood using KingFisher
Duo with 12-pin magnet head and Microtiter deep well 96 plate ..........................................................13
Protocol E. Instructions for genomic DNA purification from 1 mL of whole blood using KingFisher
Duo with 6-pin magnet head and 24 deep well plates ...........................................................................15
Protocol F. Instructions for genomic DNA purification from 2 mL of whole blood using KingFisher
Duo with 6-pin magnet head and 24 deep well plates ...........................................................................17
Protocol G. Instructions for manual genomic DNA purification from 200 μL of whole blood .............19
Protocol H. Instructions for DNA purification from dried blood .............................................................20
Protocol I. Instructions for DNA purification from buffy coat .................................................................20
Protocol J. Instructions for DNA purification from bone marrow ...........................................................20
Protocol K. Instructions for DNA purification from urine ........................................................................21
Protocol L. Instructions for DNA purification from swabs ......................................................................21
SAFETY INFORMATION .................................................................................................................... 22
3
COMPONENTS OF THE KIT
MagJET Whole Blood Genomic DNA Kit
Proteinase K
Lysis Buffer for MagJET Blood gDNA Kit
MagJET Magnetic Beads
Binding Buffer (conc.) for MagJET Blood gDNA Kit
Wash Buffer 1 (conc.) for MagJET Blood gDNA Kit
Wash Buffer 2 (conc.) for MagJET Blood gDNA Kit
Elution Buffer
#K2741
96 preps
2  1.0 mL
15 mL
2  1.4 mL
23 mL
100 mL
40 mL
30 mL
#K2742
384 preps
8  1.0 mL
60 mL
10.6 mL
90 mL
4  100 mL
2  40mL
3  30mL
STORAGE
Proteinase K solution is stable at room temperature as long as the vial remains sealed. After
opening, store at -20°C. MagJET Magnetic Beads should be stored at 4°C. Other components
of the kit should be stored at room temperature (15-25°C).
DESCRIPTION
The MagJET Whole Blood Genomic DNA Kit is designed for fast and efficient purification of
genomic DNA from fresh or frozen whole blood treated with EDTA or citrate, as well as blood
fractions.
Note: Samples preserved with heparin are not compatible with this kit.
The kit utilizes paramagnetic bead technology enabling high yields and robust performance.
High binding capacity, uniform particle size, and rapid magnetic response of MagJET magnetic
beads makes the technology ideal for high throughput automatic nucleic acid purification, as
well as for manual nucleic acid purification by low sample throughput users.
The resulting high quality DNA is free of proteins, nucleases and other contaminants or
inhibitors, can be used in a wide range of downstream applications such as PCR, qPCR and
other enzymatic reactions.
PRINCIPLE
The MagJET Whole Blood Genomic DNA Kit uses a highly efficient MagJET magnetic particlebased technology for nucleic acid purification. The whole nucleic acid isolation process
combines simple steps of sample lysis, DNA binding to the magnetic beads, washing and
elution.
Purification protocols optimized for automated KingFisher instruments utilize a high throughput
magnetic bead transfer technique where magnetic beads are transferred through different
reagent plates containing lysis, binding, wash and elution reagents. This enables high
throughput nucleic acid purification and eliminates multiple pipetting steps.
Alternatively protocol is available where instead of magnetic particles, buffers and other
reagents are transferred in each of the protocol steps, while magnetic beads remain captured
on the wall of the tube with the help of a magnetic rack. This allows the kit to be used in
various throughput applications using a magnetic rack and manual or automated pipetting
equipment.
4
IMPORTANT NOTES
 Add the indicated volumes of ethanol (96-100%) to Binding Buffer, concentrated Wash
Buffer 1 and 2 prior to first use:
Binding
Buffer
96 preps
Wash
Buffer 1
Wash
Buffer 2
Binding
Buffer
384 preps
Wash
Buffer 1
Wash
Buffer 2
Concentrated
23 mL
100 mL
40 mL
90 mL
100 mL
40 mL
buffer
Ethanol
23 mL
100 mL
200 mL
90 mL
100 mL
200 mL
(96-100%)
Total volume:
46 mL
200 mL
240 mL
180 mL
200 mL
240 mL
After preparing each solution, mark the bottle to indicate that this step has been completed.
 Check all solutions in the kit for any salt precipitation before each use. Re-dissolve any
precipitates by warming the solution at 37°C, and then equilibrate to room temperature (1525°C).
 Wear gloves when handling the Lysis Buffer, Binding Buffer and Wash Buffer 1 as these
reagents contain irritants (see page 22 for SAFETY INFORMATION).






ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED
Pipettes and pipette tips
1.5 mL tubes
Disposable gloves
96-100% ethanol, molecular biology grade
Automatic magnetic particle processor and consumables or
Magnetic particle processing rack.
5
PROTOCOL SELECTION GUIDE
The MagJET Whole Blood Genomic DNA Kit provides optimized protocols for genomic DNA
purification from different amounts of starting material (200 µL, 1 mL and 2 mL). The kit is
compatible with automated and manual sample processing, allowing low- to high-throughput
nucleic acid purification workflow.
The following selection guide summarizes available protocols depending on starting sample
volume, throughput and sample processing type. Automation protocols are optimized for
KingFisher Flex and KingFisher Duo instruments.
Throughput
per run
KingFisher Flex
Instrument
KingFisher Duo
Instrument
Manual
processing
Protocol selection guide:
MagJET
purification
protocol
96
●
-
-
Protocol A
page 7
12
-
●
-
Protocol D
page 13
24
●
-
-
Protocol B
page 9
6
-
●
-
Protocol E
page 15
24
●
-
-
Protocol C
page 11
6
-
●
-
Protocol F
page 17
200 µL
variable
-
-
●
Protocol G
page 19
1.5 mL of
whole
blood
20 µL
variable
●
●
●
Protocol H
page 20
variable
●
●
●
Protocol I
page 20
variable
●
●
●
Protocol J
page 20
Urine
0.5 mL
variable
●
●
●
Protocol K
page 21
Swabs
-
variable
●
●
●
Protocol L
page 21
Sample type
Sample
volume
200 µL
Fresh or frozen
whole blood
treated with
EDTA or
citrate.
Dried blood
Buffy coat
Bone marrow
1 mL
2 mL
6
Page
GENOMIC DNA PURIFICATION PROTOCOLS AND PIPETTING INSTRUCTIONS
Protocols A-G (pages 7-19) are recommended for genomic DNA purification from fresh or
frozen whole blood treated with EDTA or citrate. Note: Samples preserved with heparin are
not compatible with the kit. For genomic DNA purification from dried blood, buffy coat, bone
marrow, urine or swabs follow Protocols H-L (pages 20-21).
Protocol A. Instructions for genomic DNA purification from 200 μL of whole blood using
KingFisher Flex 96 and Microtiter deep well 96 plates
Note:
 When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time,
prepare working solutions of Wash Buffer 1, Wash Buffer 2 and Binding Buffer as described
on page 5.
 Transfer the Blood_gDNA_200uL_Flex protocol file to the KingFisher instrument before
first use. The instructions for transferring the protocol can be found in Chapter 4: “Using the
software” in the BindIt Software for KingFisher Instruments version 3.1 User Manual. The
protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web
page on www.thermoscientific.com/onebio
1. Obtain four empty Thermo Scientific Microtiter deep well 96 plates and two empty Thermo
Scientific KingFisher Flex 96 KF plates.
2. Prepare the Sample plate (Microtiter deep well 96 plate).
Add the following reagents to the Sample plate and leave the plate at room temperature while
the other plates are being filled.
Plate
number
1
Plate type
Microtiter deep well 96 plate
Plate name
Sample
Content
Sample/reagent
volume per well
Blood sample
200 µL
Lysis Buffer
100 µL
Proteinase K
20 µL
Content
Sample/reagent
volume per well
3. Prepare the other plates as follows:
Plate
number
Plate type
Plate name
2
Microtiter deep well 96 plate Wash 1_1
Wash Buffer 1
800 µL
3
Microtiter deep well 96 plate Wash 1_2
Wash Buffer 1
800 µL
4
Microtiter deep well 96 plate
Wash 2
Wash Buffer 2
1000 µL
5
KingFisher Flex 96 KF plate
Elution
Elution Buffer
150 µL
6
KingFisher Flex 96 KF plate
Tip plate
4. Place a Thermo Scientific KingFisher Flex 96 tip comb for deep well magnets on an empty
Tip plate KingFisher Flex 96 KF plate).
5. Start the Blood_gDNA_200uL_Flex protocol with the KingFisher Flex 96 and load the
plates according to the instructions on the KingFisher display. After all the plates have been
loaded into the instrument, the protocol will begin.
7
6. Add the MagJET Magnetic Bead suspension resuspended well by vortexing and
Binding Buffer (supplemented with ethanol, see page 5) to the Sample plate, when the
KingFisher Flex pauses at the dispense step after the lysis step (approximately 5 minutes
after starting the protocol run).
Plate
number
Plate type
Plate name
1
Microtiter deep well 96 plate
Sample
Content
Sample/reagent
volume per well
Magnetic beads
25 µL
Binding Buffer
400 µL
7. Place the Sample plate back into the instrument and press Start. After the pause, the
protocol will continue to the end.
8. When the protocol is completed, remove the plates according to the instructions on the
KingFisher Flex display and turn off the instrument. Use the purified DNA immediately in
downstream applications or store at -20°C.
8
Protocol B. Instructions for genomic DNA purification from 1 mL of whole blood using
KingFisher Flex and 24 deep well plates.
Note:
 When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time,
prepare working solutions of Wash Buffer 1, Wash Buffer 2 and Binding Buffer as described
on page 5.
 Transfer the Blood_gDNA_1mL_Flex protocol file to the KingFisher instrument before first
use. The instructions for transferring the protocol can be found in Chapter 4: “Using the
software” in the BindIt Software for KingFisher Instruments version 3.1 User Manual. The
protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web
page on www.thermoscientific.com/onebio
1. Obtain five empty Thermo Scientific KingFisher Flex 24 deep well plates and Tip plate.
2. Prepare the Sample plate (KingFisher Flex 24 deep well plate).
Add the following reagents to the Sample plate and leave the plate at room temperature while
the other plates are being filled
Plate
number
Plate type
1
KingFisher Flex 24 deep
well plate
Content
Sample/reagent
volume per well
Blood sample
1000 µL
Lysis Buffer
500 µL
Proteinase K
60 µL
Plate name
Content
Sample/reagent
volume per well
Wash 1_1
Wash Buffer 1
3700 µL
Wash 1_2
Wash Buffer 1
3700 µL
Wash 2
Wash Buffer 2
3700 µL
Elution
Elution Buffer
900 µL
Plate name
Sample
3. Fill the other plates as follows.
Plate
number
Plate type
2
3
4
5
KingFisher Flex 24 deep
well plate
4. Place a Thermo Scientific KingFisher Flex 24 tip comb on a Tip plate (empty KingFisher
Flex 24 deep well plate).
5. Start the Blood_gDNA_1mL_Flex protocol with the KingFisher Flex 24 and load the plates
according to the instructions on the KingFisher display. After all the plates have been
loaded into the instrument, the protocol will begin.
6. Add the MagJET Magnetic Beads resuspended well by vortexing and Binding Buffer
(supplemented with ethanol, see page 5) to the Sample plate, when the KingFisher Flex
pauses at the dispense step after the lysis step (approximately 10 minutes after starting the
protocol run).
9
Plate
number
Plate type
Plate name
1
KingFisher Flex 24
deep well plate
Sample
Content
Sample/reagent
volume per well
Magnetic beads
70 µL
Binding Buffer
1600 µL
7. Place the Sample plates back into the instrument and press Start. After the pause, the
protocol will continue to the end.
8. When the protocol is completed, remove the plates according to the instructions on the
KingFisher display and turn off the instrument. Transfer the eluate (which contains the
purified DNA) to a new clean tube, and then close immediately. Use the purified DNA
immediately in downstream applications or store at -20°C.
10
Protocol C. Instructions for DNA purification from 2 mL of whole blood using
KingFisher Flex and 24 deep well plates
Note:
 When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time,
prepare working solutions of Wash Buffer 1, Wash Buffer 2 and Binding Buffer as described
on page 5.
 Transfer the Blood_gDNA_2mL_Flex protocol file to the KingFisher instrument before first
use. The instructions for transferring the protocol can be found in Chapter 4: “Using the
software” in the BindIt Software for KingFisher Instruments version 3.1 User Manual. The
protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web
page on www.thermoscientific.com/onebio
1. Obtain six empty Thermo Scientific KingFisher Flex 24 deep well plates and a Tip plate.
2. Prepare the Sample plate 1 and 2 (KingFisher Flex 24 deep well plates). The 2 mL blood
sample is divided into two different plates, Sample plate 1 and 2. Pipette 1 mL sample into
the well on Sample plate 1 and 1 mL sample into the corresponding well on Sample
plate 2. For example, a 2 mL blood sample is divided, into wells A1 on Sample plate 1 and
A1 on Sample plate 2. Add the following reagents to the Sample plates 1 and 2, and leave
the plates at room temperature while the other plates are being filled.
Plate
number
Plate type
1
Plate name
Sample 1
Sample/reagent
volume per well
Blood sample
1000 µL
Lysis Buffer
500 µL
Proteinase K
60 µL
Blood sample
1000 µL
Lysis Buffer
500 µL
Proteinase K
60 µL
Plate name
Content
Sample/reagent
volume per well
Wash 1_1
Wash Buffer 1
4000 µL
Wash 1_2
Wash Buffer 1
4000 µL
Wash 2
Wash Buffer 2
4000 µL
Elution
Elution Buffer
900 µL
KingFisher Flex 24 deep
well plate
2
Content
Sample 2
3. Fill the other plates as follows:
Plate
number
Plate type
3
4
5
6
KingFisher Flex 24 deep
well plate
4. Place a Thermo Scientific KingFisher Flex 24 tip comb on a Tip plate (KingFisher Flex 24
deep well plate).
5. Start the Blood_gDNA_2mL_Flex protocol with the KingFisher Flex 24 and load the plates
according to the instructions on the KingFisher display. After all the plates have been
loaded into the instrument, the protocol will begin.
11
6. Add the MagJET Magnetic Beads resuspended well by vortexing and Binding Buffer
(supplemented with ethanol, see page 5) to the Sample plates, when the KingFisher
Flex24 pauses at the dispense step after the lysis step (approximately 5 minutes after
starting the protocol run).
Plate
number
1
2
Plate type
KingFisher Flex 24
deep well plate
Plate name
Sample 1
Sample 2
Content
Sample/reagent
volume per well
Magnetic beads
70 µL
Binding Buffer
1600µL
Magnetic beads
70 µL
Binding Buffer
1600 µL
7. Place the Sample plates back into the instrument and press Start. After the pause, the
protocol will continue to the end.
8. After the run is finished, remove the plates and transfer the eluate (which contains the
purified DNA) to a clean and new tube, then close immediately.
When the protocol is completed, remove the plates according to the instructions on the
KingFisher display and turn off the instrument. Transfer the eluate (which contains the purified
DNA) to a new clean tube, and then close immediately. Use the purified DNA immediately in
downstream applications or store at -20°C.
12
Protocol D. Instructions for genomic DNA purification from 200 μL of whole blood using
KingFisher Duo with 12-pin magnet head and Microtiter deep well 96 plate
Note:
 When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time,
prepare working solutions of Wash Buffer 1, Wash Buffer 2 and Binding Buffer as described
on page 5.
 Transfer the Blood_gDNA_200uL_Duo protocol file to the KingFisher instrument before
first use. The instructions for transferring the protocol can be found in Chapter 4: “Using the
software” in the BindIt Software for KingFisher Instruments version 3.1 User Manual. The
protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web
page on www.thermoscientific.com/onebio
 Ensure you are using the KingFisher Duo 12-pin magnet head and heating block.
1. Obtain one empty Thermo Scientific Microtiter 96 deep well plate and one Thermo Scientific
KingFisher Duo elution strip.
2. Prepare the Blood DNA plate (Microtiter deep well 96 plate).
Add the following reagents to the rows. Note that row B is reserved for the tip and should be
left empty. Note that rows C, D and H are left empty.
Plate name and type
Blood DNA plate
Microtiter deep well 96 plate
Row
Row name
A
Sample
B
C
D
E
F
G
H
Tip
Empty
Empty
Wash 1_1
Wash 1_2
Wash 2
Empty
Content
Blood sample
Lysis Buffer
Proteinase K
12-tip comb
Empty
Empty
Wash Buffer 1
Wash Buffer 1
Wash Buffer 2
Empty
Sample/reagent
volume per well
200 µL
100 µL
20 µL
Empty
Empty
Empty
800 µL
800 µL
1000 µL
Empty
3. Fill the KingFisher Duo elution strip as follows. Make sure that the elution strip is placed
in the correct direction into the elution block. Ensure that the perforated end is facing
towards the user and the Elution Buffer is pipetted into the correct wells.
Elution strip
KingFisher Duo elution strip
Content
Elution Buffer
Reagent volume per well
130 µL
4. Place a Thermo Scientific KingFisher Duo 12-tip comb into row B on the Blood DNA plate.
5. Switch on the KingFisher Duo instrument and ensure you are using the KingFisher Duo
12-pin magnet head and heating block. Start the Blood_gDNA_200uL_Duo protocol. Insert
the Blood DNA plate and elution strip into the instrument as indicated on the KingFisher
Duo display. Make sure that the elution strip is placed in the correct direction into the elution
block. Ensure that the perforated end is facing towards the user.
13
6. Add the MagJET Magnetic beads resuspended well by vortexing and Binding Buffer
(supplemented with ethanol, see page 5) to row A of the Blood DNA plate.
Plate name and type
Blood DNA plate
Microtiter deep well 96 plate
Row
Row name
Content
A
Sample
Magnetic beads
Binding Buffer
Sample/reagent
volume per well
25 µL
400 µL
7. Place the Blood DNA plate back into the instrument and press Start. After the pause, the
protocol will continue to the end.
8. When the protocol is complete, remove the plate and elution strip according to the
instructions on the KingFisher Duo display and turn off the instrument. Transfer the eluate
(which contains the purified DNA) to a new clean tube, and then close immediately. The
purified DNA is ready for use in downstream applications.
14
Protocol E. Instructions for genomic DNA purification from 1 mL of whole blood using
KingFisher Duo with 6-pin magnet head and 24 deep well plates
Note:
 When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time,
prepare working solutions of Wash Buffer 1, Wash Buffer 2 and Binding Buffer as described
on page 5.
 Transfer the Blood_gDNA_1mL_Duo protocol file to the KingFisher instrument before first
use. The instructions for transferring the protocol can be found in Chapter 4: “Using the
software” in the BindIt Software for KingFisher Instruments version 3.1 User Manual. The
protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web
page on www.thermoscientific.com/onebio
 Ensure that you are using the KingFisher Duo 6-pin magnet head and heating block.
1. Obtain two empty Thermo Scientific KingFisher Flex 24 deep well plates.
2. Prepare the Blood DNA plates 1 and 2. Add the following reagents to the Blood DNA
plates 1 and 2 (KingFisher Flex 24 deep well plates). Pipette 1 mL sample into the wells of
row A of the Blood DNA plate 1.
Add the following reagents to the rows. Note that row B of the Blood DNA plate 1 is reserved
for the tip and should be left empty. Note that rows C of the Blood DNA plate 1 and D of the
Blood DNA plate 2 are left empty too.
Plate name and type
Blood DNA plate 1
KingFisher Flex 24 deep well
plate
Blood DNA plate 2
KingFisher Flex 24 deep well
plate
Row
Row name
A
Sample
B
C
D
A
B
C
D
Tip
Empty
Wash 1_1
Elution
Wash 2
Wash 1_2
Empty
Content
Blood sample
Lysis Buffer
Proteinase K
12-tip comb
Empty
Wash Buffer 1
Elution Buffer
Wash Buffer 2
Wash Buffer 1
Empty
Sample/reagent
volume per well
1000 µL
500 µL
80 µL
Empty
Empty
3500 µL
900 µL
5000 µL
3500 µL
Empty
3. Place a Thermo Scientific KingFisher Duo 6-tip comb into row B of the Blood DNA plate 1.
4. Switch on the KingFisher Duo and make sure that you are using the KingFisher Duo 6-pin
magnet head and heating block. Start the Blood_gDNA_1mL_Duo protocol. Insert the
Blood DNA plates into the instrument as indicated on the KingFisher Duo display. After
both plates have been loaded into the instrument, the protocol will begin.
5. Add the MagJET Magnetic beads resuspended well by vortexing and Binding Buffer
(supplemented with ethanol, see page 5) to row A of the Blood DNA plate 1, when the
KingFisher Duo pauses at the dispense step after the lysis step (approximately 10 minutes
after starting the protocol run).
15
Plate name and type
Blood DNA plate 1
KingFisher Flex 24 deep well plate
Row
Row name
A
Sample
Sample/reagent
volume per well
Magnetic beads
100 µL
Binding Buffer
1840 µL
Content
6. Place the Blood DNA plate 1 back into the instrument and press Start. After the pause, the
protocol will continue to the end.
7. When the protocol is complete, remove the plate and elution strip according to the
instructions on the KingFisher Duo display and turn off the instrument. Transfer the eluate
(which contains the purified DNA) to a new clean tube, and then close immediately. The
purified DNA is ready for use in downstream applications.
16
Protocol F. Instructions for genomic DNA purification from 2 mL of whole blood using
KingFisher Duo with 6-pin magnet head and 24 deep well plates
Note:
 When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time,
prepare working solutions of Wash Buffer 1, Wash Buffer 2 and Binding Buffer as described
on page 5.
 Transfer the Blood_gDNA_2mL_Duo protocol file to the KingFisher instrument before first
use. The instructions for transferring the protocol can be found in Chapter 4: “Using the
software” in the BindIt Software for KingFisher Instruments version 3.1 User Manual. The
protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web
page on www.thermoscientific.com/onebio
 Ensure that you are using the KingFisher Duo 6-pin magnet head and heating block
1. Obtain two empty Thermo Scientific KingFisher Flex 24 deep well plates.
2. Prepare the Blood DNA plates 1 and 2 by adding the following reagents to the Blood DNA
plates 1 and 2 (KingFisher Flex 24 deep well plates). The 2 mL blood sample is divided into
two different wells of rows A and B of the Blood DNA plate 1. Pipette 1 mL sample into the
well of row A and 1 mL sample into corresponding well of row B. A 2 mL blood sample is
divided, for example, into wells A1 and B1 of the Blood DNA plate 1.
3. Add the following reagents to the rows. Note that row C of the Blood DNA plate 1 is
reserved for the tip and should be left empty.
Plate name and type
Blood DNA plate 1
KingFisher Flex 24 deep well
plate
Blood DNA plate 2
KingFisher Flex 24 deep well
plate
Row
Row name
A
Sample 1
B
Sample 2
C
D
A
B
C
D
Tip
Wash 1_1
Elution
Wash 2_2
Wash 2_1
Wash 1_2
Content
Blood sample
Lysis Buffer
Proteinase K
Blood sample
Lysis Buffer
Proteinase K
12-tip comb
Wash Buffer 1
Elution Buffer
Wash Buffer 2
Wash Buffer 2
Wash Buffer 1
Sample/reagent
volume per well
1000 µL
500 µL
60 µL
1000 µL
500 µL
60 µL
Empty
4800 µL
900 µL
5000 µL
5000 µL
4800 µL
4. Place a Thermo Scientific KingFisher Duo 6-tip comb into row C of the Blood DNA plate 1.
5. Switch on the KingFisher Duo and make sure that you are using the KingFisher Duo 6-pin
magnet head and heating block. Start the Blood_gDNA_2mL_Duo protocol. Insert the
Blood DNA plates into the instrument as indicated on the KingFisher Duo display. After
both plates have been loaded into the instrument, the protocol will begin.
17
6. Add the MagJET Magnetic beads resuspended well by vortexing and Binding Buffer
(supplemented with ethanol, see page 5) to rows A and B of the Blood DNA plate 1,
when the KingFisher Flex pauses at the dispense step after the lysis step (approximately
10 minutes after starting the protocol run).
Plate name and type
Blood DNA plate 1
KingFisher Flex 24 deep well
plate
Row
Row name
A
Sample 1
B
Sample 1
Content
Magnetic beads
Binding Buffer
Magnetic beads
Binding Buffer
Sample/reagent
volume per well
70 µL
1600 µL
70 µL
1600 µL
7. Place the Blood DNA plate 1 back into the instrument and press OK. After the pause, the
protocol will continue to the end.
8. When the protocol is complete, remove the plates according to the instructions on the
KingFisher Duo display and turn off the instrument. Transfer the eluate (which contains the
purified DNA) to a new clean tube, and then close immediately. The purified DNA is ready
for use in downstream applications.
18
Protocol G. Instructions for manual genomic DNA purification from 200 μL of whole
blood
The following protocol is based on transfer of liquids by pipetting through different purification
steps rather than magnetic bead transfer as in KingFisher automatic protocols. This allows the
kit to be used in various throughput applications using magnetic rack and manual or automated
pipetting equipment. Protocols for the other automated pipetting platforms should be optimized
for each platform and sample used. To enable protocol optimization all buffers are available to
purchase separately.
Note: When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time,
prepare working solutions of Wash Buffer 1, Wash Buffer 2 and Binding Buffer as described on
page 5.
1. Add 20 μL Proteinase K and 100 μL Lysis Buffer to a 200 μL blood sample. Vortex 30 s
(at maximum speed) and incubate 3 minutes at room temperature (15-25°C). Vortex 15 s
to mix and briefly spin to remove drops from inside of the lid.
2. Add 25 μL MagJET Magnetic beads resuspended well by vortexing and 400 μL Binding
Buffer (supplemented with ethanol, see page 5). Resuspend the magnetic beads by
vortexing, briefly spin to remove droplets from inside of the lid. Place the tube on the
magnetic rack and let the magnetic beads collect at the magnet for 3 minutes. Discard the
supernatant by using a pipette.
3. Remove the magnetic rack and add 800 μL Wash Buffer 1 (supplemented with ethanol,
see page 5). Resuspend the magnetic beads by vortexing, briefly spin to remove drops from
inside of the lid. Place the tube on the magnetic rack and let the magnetic beads collect at
the magnet for 2-3 minutes. Discard the supernatant by using a pipette.
4. Repeat step 3 using 800 μL of Wash Buffer 1.
5. Remove the magnetic rack and add 1000 μL Wash Buffer 2 (supplemented with ethanol,
see page 5). Resuspend the magnetic beads by vortexing, briefly spin to remove drops from
inside of the lid. Place the tube on the magnetic rack and let the magnetic beads collect at
the magnet for 2-3 minutes. Discard the supernatant by using a pipette. Carefully eliminate
supernatant droplets using a small pipette tip and make sure no droplets are left on the tube
wall.
Note: Make sure that all the supernatant is completely removed in the last washing step. If
there are any visible droplets incubate at room temperature (15-25°C) for 1 minute.
6. Remove the magnetic rack and add 150 μL Elution Buffer. Resuspend the magnetic beads
by vortexing, incubate for 5 minutes at 72°C, place the tube on the magnetic rack and let
the magnetic beads collect at the magnet for 2-3 minutes.
7. While on the magnetic rack, transfer the eluate (which contains the purified DNA) to a new
clean tube, then close immediately. Use the purified DNA immediately in downstream
applications or store at -20°C.
19
Protocol H. Instructions for DNA purification from dried blood
1
2
3
Cut out the section of filter containing the dried blood sample and place into a
microcentrifuge tube.
Add 200 μL of 0.9% (w/v) NaCl and incubate 5-10 minutes at room temperature
(15-25°C).
For manual purification, proceed to Step 1 of the Protocol G “Instructions for
manual genomic DNA purification from 200 μL of whole blood” on page 19.
For automated purification using KingFisher Flex 96 or KingFisher Duo Instruments
proceed to Step 1 of corresponding Protocol A “Instructions for genomic DNA
purification from 200 μL of whole blood using KingFisher Flex 96 and Microtiter
deep well 96 plates” on page 7 or Protocol D “Instructions for genomic DNA
purification from 200 μL of whole blood using KingFisher Duo with 12-pin
magnet head and 96 deep well plate” on page 13.
Protocol I. Instructions for DNA purification from buffy coat
1
2
3
4
Centrifuge 1.5 mL of whole blood at 2,500 × g (~5,000 rpm) for 10 minutes at room
temperature. Three layers should be visible.
Remove upper clear layer by aspiration.
Collect approximately 200 μL of intermediate layer using an automatic pipette.
For manual purification, proceed to Step 1 of the Protocol G “Instructions for
manual genomic DNA purification from 200 μL of whole blood” on page 19.
For automated purification using KingFisher Flex 96 or KingFisher Duo Instruments
proceed to Step 1 of corresponding Protocol A “Instructions for genomic DNA
purification from 200 μL of whole blood using KingFisher Flex 96 and Microtiter
deep well 96 plates” on page 7 or Protocol D “Instructions for genomic DNA
purification from 200 μL of whole blood using KingFisher Duo with 12-pin
magnet head and 96 deep well plate” on page 13.
Protocol J. Instructions for DNA purification from bone marrow
1
2
Harvest 20 μL of fresh or frozen bone marrow.
For manual purification, proceed to Step 1 of the Protocol G “Instructions for
manual genomic DNA purification from 200 μL of whole blood” on page 19.
For automated purification using KingFisher Flex 96 or KingFisher Duo Instruments
proceed to Step 1 of corresponding Protocol A “Instructions for genomic DNA
purification from 200 μL of whole blood using KingFisher Flex 96 and Microtiter
deep well 96 plates” on page 7 or Protocol D “Instructions for genomic DNA
purification from 200 μL of whole blood using KingFisher Duo with 12-pin
magnet head and 96 deep well plate” on page 13.
20
Protocol K. Instructions for DNA purification from urine
1
2
3
4
5
Add 0.5 mL of 0.5 M EDTA to 4.5 mL of urine (final concentration 50 mM).
Centrifuge 10 minutes at 800 × g (~3,000 rpm).
Discard the supernatant.
Resuspend the pellet in 200 μL of 0.9% (w/v) NaCl.
For manual purification, proceed to Step 1 of the Protocol G “Instructions for
manual genomic DNA purification from 200 μL of whole blood” on page 19.
For automated purification using KingFisher Flex 96 or KingFisher Duo Instruments
proceed to Step 1 of corresponding Protocol A “Instructions for genomic DNA
purification from 200 μL of whole blood using KingFisher Flex 96 and Microtiter
deep well 96 plates” on page 7 or Protocol D “Instructions for genomic DNA
purification from 200 μL of whole blood using KingFisher Duo with 12-pin
magnet head and 96 deep well plate” page 13.
Protocol L. Instructions for DNA purification from swabs
1
2
3
To collect a sample, scrape the swab 5-6 times against the inside cheek.
Swirl the swab for 30-60 s in 200 μL of 0.9% (w/v) NaCl.
For manual purification, proceed to Step 1 of the Protocol G “Instructions for manual
genomic DNA purification from 200 μL of whole blood” on page 19.
For automated purification using KingFisher Flex 96 or KingFisher Duo Instruments
proceed to Step 1 of corresponding Protocol A “Instructions for genomic DNA
purification from 200 μL of whole blood using KingFisher Flex 96 and Microtiter
deep well 96 plates” on page 7 or Protocol D “Instructions for genomic DNA
purification from 200 μL of whole blood using KingFisher Duo with 12-pin magnet
head and 96 deep well plate” page 13.
21
SAFETY INFORMATION
Lysis Buffer for MagJET Blood gDNA Kit
Xn Harmful
Hazard-determining components of labelling: guanidinium chloride
Risk phrases:
22
Harmful if swallowed.
36/38 Irritating to eyes and skin.
Safety phrases:
23
Do not breathe gas/fumes/vapour/spray.
26
In case of contact with eyes, rinse immediately with plenty of water and seek medical
advice.
36/37 Wear suitable protective clothing and gloves.
60
This material and its container must be disposed of as hazardous waste.
Binding Buffer for MagJET Blood gDNA Kit
Xn Harmful
O Oxidizing
Hazard-determining components of labelling: sodium perchlorate
Risk phrases:
22
Harmful if swallowed.
9
Explosive when mixed with combustible material.
Safety phrases:
17
Keep away from combustible material.
23
Do not breathe gas/fumes/vapour/spray.
27
Take off immediately all contaminated clothing.
36
Wear suitable protective clothing.
60
This material and its container must be disposed of as hazardous waste.
Wash Buffer 1 (conc.) for MagJET Blood gDNA Kit
O Oxidizing
Hazard-determining components of labelling: sodium perchlorate
Risk phrases:
9
Explosive when mixed with combustible material.
Safety phrases:
17
Keep away from combustible material.
27
Take off immediately all contaminated clothing.
60
This material and its container must be disposed of as hazardous waste.
22
Proteinase K
Xn Harmful
Hazard-determining components of labelling: Proteinase, Tritirachium album serine
Risk phrases:
42
May cause sensitisation by inhalation.
Safety phrases:
23
Do not breathe gas/fumes/vapour/spray.
36
Wear suitable protective clothing.
45
In case of accident or if you feel unwell, seek medical advice immediately (show the
label where possible).
60
This material and its container must be disposed of as hazardous waste.
PRODUCT USE LIMITATION
This product is developed, designed and sold exclusively for research purposes and in vitro use only. The
product was not tested for use in diagnostics or for drug development, nor is it suitable for administration to
humans or animals.
Please refer to www.thermoscientific.com/onebio for Material Safety Data Sheet of the product.
© 2013 Thermo Fisher Scientific, Inc. All rights reserved. All (other) trademarks are the property of Thermo
Fisher Scientific, Inc. and its subsidiaries.
23